Telmisartán y losartán: las marcadas diferencias entre sus propiedades químicas y farmacológicas pueden explicar la diferencia en la eficacia terapéutica en pacientes hospitalizados con covid-19 / Telmisartan and losartan: marked differences in chemical and pharmacological properties may explain differences in therapeutic efficacy in hospitalized patients with COVID-19

El objetivo de este artículo es comparar las propiedades químicas y farmacológicas del telmisartán y el losartán, y su metabolito activo EXP3174, con el fin de entender por qué el telmisartán es efectivo en pacientes hospitalizados con Covid-19 mientras que el losartán no lo es. Se llevó a cabo una revisión bibliográfica exhaustiva de las propiedades químicas, farmacocinéticas y farmacodinámicas de ambos fármacos y se destacaron las diferencias más importantes que podrían estar relacionadas con su efectividad en pacientes con Covid-19. Se concluyó que las propiedades farmacológicas del telmisartán, como su mayor afinidad por el receptor AT1, su duración de acción prolongada y su capacidad para modular la inflamación podrían explicar su efectividad en pacientes con Covid-19. Por otro lado, las propiedades farmacológicas del losartán, como su menor afinidad por el receptor AT1 y su rápido metabolismo, pueden limitar su efectividad en pacientes con Covid-19. Estos resultados resaltan la importancia de comprender las propiedades químicas y farmacológicas de los medicamentos para identificar posibles candidatos terapéuticos efectivos en el tratamiento de Covid-19. (AU)

The objective of this article is to compare the chemical and pharmacological properties of telmisartan and losartan and their active metabolite EXP3174 to understand why telmisartan is effective in hospitalized patients with COVID-19 while losartan is not. A comprehensive literature review of the chemical, pharmacokinetic and pharmacodynamic properties of both drugs was done to highlight the most important differences that may be related to their efficacy in patients with COVID-19. It was concluded that the pharmacological properties of telmisartan, such as its higher affinity for the AT1 receptor, its long duration of action and its ability to modulate inflammation, could explain its efficacy in patients with COVID-19. On the other hand, the pharmacological properties of losartan, such as its lower affinity for the AT1 receptor and its rapid metabolism, may limit its efficacy in patients with COVID-19. These results highlight the importance of understanding the chemical and pharmacological properties of drugs to identify potential effective therapeutic candidates for the treatment of COVID-19. (AU)

Angiotensin Receptor Blockers for COVID-19: Pathophysiological and Pharmacological Considerations About Ongoing and Future Prospective Clinical Trials.

COVID-19 pandemic demands a swift response to find therapeutic tools that effectively reduce morbidity and mortality. Despite initial fears, evidence from retrospective observational studies supports the inhibition of the renin-angiotensin system as an emerging pathway to delay or moderate angiotensin II-driven lung inflammation. This has triggered several prospective clinical trials. In this commentary we provide an overview and analysis of current ongoing clinical trials aimed at evaluating the therapeutic efficacy of angiotensin receptor blocker (ARB) use in COVID-19. The relevance of the results of these trials will have to be interpreted depending on the stage and severity of the disease and in light of the start time of their prescription related to the time of diagnosis of COVID-19 as well as the administered doses.

Telmisartan as tentative angiotensin receptor blocker therapeutic for COVID-19.

In late 2019, a new coronavirus emerged in Wuhan Province, China, causing lung complications similar to those produced by the SARS coronavirus in the 2002-2003 epidemic. This new disease was named COVID-19 and the causative virus SARS-CoV-2. The SARS-CoV-2 virus enters the airway and binds, by means of the S protein on its surface to the membrane protein ACE2 in type 2 alveolar cells. The S protein-ACE2 complex is internalized by endocytosis leading to a partial decrease or total loss of the enzymatic function ACE2 in the alveolar cells and in turn increasing the tissue concentration of pro-inflammatory angiotensin II by decreasing its degradation and reducing the concentration of its physiological antagonist angiotensin 1-7. High levels of angiotensin II on the lung interstitium can promote apoptosis initiating an inflammatory process with release of proinflammatory cytokines, establishing a self-powered cascade, leading eventually to ARDS. Recently, Gurwitz proposed the tentative use of agents such as losartan and telmisartan as alternative options for treating COVID-19 patients prior to development of ARDS. In this commentary article, the authors make the case for the election of telmisartan as such alternative on the basis of its pharmacokinetic and pharmacodynamic properties and present an open-label randomized phase II clinical trial for the evaluation of telmisartan in COVID-19 patients (NCT04355936).

Involvement of Extracellular Signal-Regulated Kinase 5 in Kinin B1 Receptor Upregulation in Isolated Human Umbilical Veins.

The upregulated kinin B1 receptors exert a pivotal role in modulating inflammatory processes. In isolated human umbilical veins (HUVs), kinin B1 receptor is upregulated as a function of in vitro incubation time and proinflammatory stimuli. The aim of this study was to evaluate, using functional and biochemical methods, the involvement of extracellular signal-regulated kinase 5 (ERK5), p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) on the kinin B1 receptor upregulation process in HUV. Real-time polymerase chain reaction analysis revealed for the first time that kinin B1 receptor mRNA expression closely parallels the functional sensitization to kinin B1 receptor selective agonist des-Arg(10)-kallidin (DAKD) in HUV. Moreover, the selective inhibition of ERK5, p38 MAPK, and JNK, but not ERK1/2, produced a dose-dependent rightward shift of the concentration-response curves to DAKD after 5-hour incubation and a reduction in kinin B1 receptor mRNA expression. Biochemical analyses showed that ERK5, p38 MAPK, and JNK phosphorylation is maximal during the first 2 hours postisolation, followed by a significant reduction in the last 3 hours. None of the treatments modified the responses to serotonin, an unrelated agonist, suggesting a specific effect on kinin B1 receptor upregulation. The present work provides for the first time pharmacologic evidence indicating that ERK5 plays a significant role on kinin B1 receptor upregulation. Furthermore, we confirm the relevance of p38 MAPK and JNK as well as the lack of effect of ERK1/2 in this process. This study may contribute to a better understanding of MAPK involvement in inflammatory and immunologic diseases.

[Pharmacogenetics in psychiatry: how far are we from clinical application?]. / Farmacogenética en psiquiatría: ¿qué tan lejos estamos de su aplicación clínica?

Pharmacogenetics studies how genetic variation influences the response of patients to drugs. This discipline has a greater impact in those medical specialties that treat complex diseases in which the therapeutic response is insufficient and/or have high costs such as psychiatry. This is a narrative review in which we analyze the main results of pharmacogenetic studies performed with the most relevant groups of psychoactive drugs and discusses missing for incorporating these advances into our daily practice. We conclude that despite the remarkable progress in the field of Pharmacogenetics in the last 10 years, studies in psychiatry have been inconclusive and the clinical use of pharmacogenetic testing is still limited. However, there are some encouraging elements about the applicability of these tools for the improvement of psychiatric treatments.

Endothelial angiotensin-converting enzyme and neutral endopeptidase in isolated human umbilical vein: an effective bradykinin inactivation pathway.

Kinins are metabolized by metallopeptidases present in different tissues. The aim of this study was to evaluate, employing functional studies in isolated human umbilical vein, the possible participation of angiotensin-converting enzyme, neutral endopeptidase and aminopeptidase P as an inactivation pathway of bradykinin, as well as assess if the endothelial layer is involved in this process. Concentration-response curves to bradykinin were constructed after 120 min incubation period on human umbilical vein rings with and without endothelium and enzymatic inhibitors were applied 30 min before construction of concentration-response curves. The presence of endothelium was confirmed by histological studies. Bradykinin-induced contractile responses were potentiated in human umbilical vein without endothelium when compared to intact tissues. Application of captopril 1 µM (angiotensin-converting enzyme inhibitor) or phosphoramidon 10 µM (neutral endopeptidase inhibitor) induced a leftward shift of bradykinin-elicited responses in human umbilical vein with endothelium while no effect was observed in tissues denuded of endothelium under the same treatment. Exposure to apstatin 10 µM (aminopeptidase P inhibitor) did not potentiate bradykinin-induced effects in intact human umbilical vein. When angiotensin-converting enzyme and neutral endopeptidase were concomitantly inhibited, there was a higher potentiation of bradykinin-elicited responses compared to the effects observed under individual inhibition of either enzyme. Moreover, concentration-response curves to FR190997, a non-peptidic bradykinin B(2) receptor agonist, were not modified under dual enzymatic inhibition. In conclusion, our results demonstrate for the first time the functional relevance of angiotensin-converting enzyme and neutral endopeptidase, localized on the endothelial layer, acting concurrently as a bradykinin inactivating pathway in isolated human umbilical vein.

Expression and functional evidence of the prostaglandin F2alpha receptor mediating contraction in human umbilical vein.

Our purposes were to perform the pharmacological characterization of PGF(2alpha) receptor (prostanoid FP-receptor) involved in human umbilical vein contraction and confirm its expression in this tissue. Umbilical cords from healthy patients after full-term deliveries were employed. The vein was dissected out of cords and used for either isolated organ bath or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. The natural prostanoid FP-receptor agonist, PGF(2alpha), and its selective analogues, latanoprost and bimatoprost free acids are full agonists (produce more than 80% of the maximal contractile response to 5-HT) in human umbilical vein. The agonist potency (pEC(50)) order was PGF(2alpha) (6.01+/-0.05)>latanoprost free acid (5.65+/-0.07)=bimatoprost free acid (5.59+/-0.08). The contractile effects of PGF(2alpha) and latanoprost free acid were blocked competitively by the prostanoid FP-receptor antagonist, AL-8810. The antagonist potencies (pK(B)) of AL-8810 vs. PGF(2alpha) (5.93+/-0.05) and vs. latanoprost free acid (6.40+/-0.08) in human umbilical vein are in good agreement with its ability to antagonize prostanoid FP receptors of rat, mouse and human cells. In all samples, clear signal was detected for cDNA amplification of prostanoid FP receptor and the specific prostanoid FP-receptor antibody recognized a protein of approximately 64 kDa. In conclusion, taking into account the obtained functional and biochemical data, we propose for the first time that human umbilical vein express prostanoid FP-receptors and these receptors could be involved in the vasoconstriction action of PGF(2alpha) in this tissue.

The endocannabinoid anandamide inhibits kinin B1 receptor sensitization through cannabinoid CB1 receptor stimulation in human umbilical vein.

The possible inhibition of kinin B(1) receptor up-regulation by arachidonoylethanolamide (anandamide) was evaluated in isolated human umbilical vein. Anandamide and its metabolically stable analogue, R-N-(2-Hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R-(+)-methanandamide), produced a selective and dose-dependent inhibition of kinin B(1) receptor-sensitized contractile responses. The inhibitory effect of anandamide on B(1) receptor-sensitized responses failed to be modified either by 5-biphenyl-4-ylmethyl-tetrazole-1-carboxylic acid dimethylamide (LY2183240), a selective anandamide uptake inhibitor, or 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl) methanone (AM630), selective cannabinoid CB(2) receptor antagonist. However, the cannabinoid CB(1) receptor antagonist, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), abolished anandamide effects on kinin B(1) receptor sensitization. The present results provide strong pharmacological evidence indicating that endocannabinoid anandamide inhibits kinin B(1) receptor up-regulation through cannabinoid CB(1) receptor stimulation in human umbilical vein.

Potentiation of adrenaline vasoconstrictor response by sub-threshold concentrations of U-46619 in human umbilical vein: involvement of smooth muscle prostanoid TP(alpha) receptor isoform.

Considering the potential physiological, pharmacological and therapeutic relevance of synergistic interaction of thromboxane A(2) with adrenaline at postjunctional receptor sites, we examined whether sub-threshold concentrations of thromboxane A(2) mimetic U-46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha)) could amplify adrenaline-induced contraction in human umbilical vein. The receptor involved in U-46619-induced potentiation of adrenaline contractility was also investigated. Umbilical cords (n=125) from healthy patients after full-term vaginal or caesarean deliveries were employed. The vein was dissected out of cords and rings used for isolated organ bath experiments or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Presence of endothelium did not modify U-46619-induced contraction in human umbilical vein. Prostanoid TP-selective receptor antagonist, SQ-29548 (7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-[1S(1alpha,2alpha(Z),3alpha,4alpha)]-5-Heptenoic acid), inhibited U-46619-induced contraction (pA(2)=8.22+/-0.11). U-46619 sub-threshold concentrations (0.1-0.3 nM) potentiated adrenaline-vasoconstriction response in a concentration-dependent manner. SQ-29548 (0.1 microM) abolished this potentiation. Using RT-PCR, we found that human umbilical vein rings with or without endothelium express the prostanoid TP(alpha), but not the prostanoid TP(beta) receptor isoform. Western blot allowed the identification of proteins with an electrophoretic mobility (47- and 55-kDa) indistinguishable from human platelet prostanoid TP receptor, a rich source of prostanoid TP(alpha) receptor isoform. Collectively, present results demonstrate that prostanoid TP(alpha) is the major receptor isoform localized on smooth muscle cells which participate in both direct vasoconstriction and potentiating effects of U-46619 on adrenaline contractions in human umbilical vein. These results suggest that thromboxane A(2) may interact synergistically with adrenaline in pathophysiological situations that lead to an increase of its umbilical venous levels (e.g. preeclampsia associated with fetal distress) raising the possibility of vasoconstriction affecting fetal blood flow.

Functional evidence of des-Arg10-kallidin enzymatic inactivating pathway in isolated human umbilical vein.

It has been known for many years that plasma and tissues contain a variety of enzymes capable of metabolizing kinins. The aim of the present study was to evaluate, by means of functional studies in a capacitance vessel such as the human umbilical vein (HUV), the possible role played by the metallopeptidases angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), and aminopeptidase M (APM) as an inactivating pathway of the B(1) receptor endogenous agonist des-Arg(10)-kallidin (DAKD). In HUV rings with and without endothelium, concentration-response curves (CRCs) to DAKD were determined after a 300-min incubation period, and enzymatic inhibitors were added to the organ baths 30 min before construction of the CRC. Presence of endothelial layer was confirmed by histological studies. There was a significant leftward shift observed in control HUV rings devoid of endothelium compared with intact tissues. Exposure to 1 microM captopril (ACE inhibitor) potentiated DAKD-elicited vasoconstrictor responses in HUV rings with endothelium while no such effect was observed in tissues devoid of endothelium. Application of 10 microM amastatin (APM inhibitor) induced a leftward shift of DAKD-elicited contractile responses in HUV with and without endothelium. On the other hand, 10 microM phosphoramidon (NEP inhibitor) showed no potentiating effect in HUV rings either with or without endothelium. However, under concurrent inhibition of ACE, NEP and APM, there was a higher potentiation of DAKD-elicited contractile responses compared with the effect observed with combined inhibition of ACE and APM. Moreover, when we evaluated contractile responses induced by Sar(0)-D-Phe(8)-des-Arg(9)-BK (a metabolically protected B(1) receptor agonist), no potentiating effect was observed under triple enzymatic inhibition. In conclusion, in the present study for the first time, we demonstrated in a capacitance vessel, HUV, that metallopeptidases ACE, NEP and APM represent a relevant functional inactivation pathway of DAKD.

Neutral endopeptidase up-regulation in isolated human umbilical artery: involvement in desensitization of bradykinin-induced vasoconstrictor effects.

Previous reports show that bradykinin B(2) receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.

Involvement of endothelial thromboxane A2 in the vasoconstrictor response induced by 15-E2t-isoprostane in isolated human umbilical vein.

The present study was undertaken to evaluate the contractile response of several E- and F-ring isoprostanes (IsoP) in human umbilical vein (HUV) and to investigate the role of the endothelium on the effect of 15-E2t-IsoP, the most potent vasoconstrictor isoprostane, in human vessels. HUV rings with or without endothelium were suspended in an organ bath for recording the isometric tension in response to different agonists. The inhibitors to be evaluated were applied 30 min before the addition of the agonist. All of the compounds tested produced concentration-dependent contractions when tested on HUV rings with endothelium. Although these compounds were equieffective, significant differences were observed in their potency, with U46619 being the most potent followed by 15-E2t-IsoP > 15-E1t-IsoP = 15-F2t-IsoP > 15-F1t-IsoP = 9-epi-15-F2t-IsoP in descending rank order of potency. 15-E2t-IsoP was the most potent of the isoprostanes evaluated and, therefore, the one employed in the present study. When intact endothelium HUV rings were used, 15-E2t-IsoP-induced contraction was unaffected by the endothelin-converting enzyme inhibitor, phosphoramidon (10 microM), suggesting that short-term endothelin-1 release is not involved in this response. However, the non-selective cyclooxygenase (COX) inhibitor, indomethacin (10 and 30 microM), and the COX-2 selective inhibitor, NS-398 (3, 10 and 30 microM) produced inhibitory effects on 15-E2t-IsoP-induced contraction of HUV rings with endothelium. These results indicate that COX-derived contractile prostanoids are involved in this effect. Furthermore, the apparent pKb values estimated for indomethacin (5.5) and NS-398 (5.4) suggest that the prostanoids involved are derived from the COX-2 isoenzyme pathway. On HUV rings with endothelium, the phospholipase A2 inhibitor, oleyloxyethyl phosphorylcholine (30 and 100 microM), induced an inhibitory effect on 15-E2t-IsoP-induced contraction, suggesting that the phospholipase A2 pathway is also involved in this effect. In addition, the thromboxane A2 synthase inhibitor furegrelate (10 and 30 microM) also inhibited 15-E2t-IsoP-induced contraction of HUV rings with endothelium, indicating that thromboxane A2 is one of the contractile prostanoids involved in this response. Endothelium denudation clearly diminished the vasoconstrictor potency of 15-E2t-IsoP, demonstrating that the endothelium releases a vasoconstrictor factor in response to 15-E2t-IsoP. The absence of an inhibitory effect at the highest concentration of furegrelate (30 microM) on 15-E2t-IsoP-induced contraction of HUV rings without endothelium suggested that endothelium is the source of thromboxane A2. We conclude that prostanoids derived from the COX-2 isoenzyme pathway participate in 15-E2t-IsoP-induced vasoconstriction of isolated HUV rings. Our results also indicate that endothelial thromboxane A2 is one of the prostanoids involved in this effect.

Pharmacological characterization of muscarinic receptor subtypes mediating vasoconstriction of human umbilical vein.

The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration-response curves were constructed for acetylcholine (ACh) (pEC50: 6.16+/-0.04; maximum response 80.00+/-1.98% of the responses induced by serotonin 10 microM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso-OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC50 6.33+/-0.03; double inhibition: pEC50 6.57+/-0.05). Atropine, nonselective muscarinic receptors antagonist, inhibited ACh-induced contraction (pKB 9.67). The muscarinic receptors antagonists pirenzepine (M1), methoctramine (M2) and pFHHSiD (M3) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M4 selective muscarinic receptors antagonist) was ineffective against ACh-induced contraction.In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh-induced responses. The M1 muscarinic receptors agonist McN-A-343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA2 for pirenzepine against McN-A-343 induced responses was 8.54. In conclusion, the data obtained in this study demonstrate the role of M1 muscarinic receptor subtypes and suggest the involvement of M3 muscarinic receptor subtypes in ACh-induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this tissue.

Human umbilical vein vasoconstriction induced by epinephrine acting on alpha1B-adrenoceptor subtype.

OBJECTIVE: Our purpose was to determine the presence of alpha(1)-adrenoceptor messenger RNA subtypes and extend the pharmacologic characterization of alpha(1)-adrenoceptors involved in human umbilical vein (HUV) contraction. STUDY DESIGN: Cords (n=124) from healthy patients after term vaginal or cesarean deliveries were used. The vein was carefully dissected out of cords and used for reverse transcription combined with polymerase chain reaction (RT-PCR) to amplify alpha(1)-adrenoceptor transcripts. In isolated organ baths, HUV rings were mounted and cumulative concentration-response curves were constructed either for epinephrine or the selective alpha(1A)-adrenoceptor agonist, A-61603. In other series of experiments, the effects of the selective alpha(1A)- and alpha(1B)-adrenoceptor antagonists (RS-100329 or B8805-033 or spiperone, AH11110A and cyclazosin, respectively) were evaluated to estimate its blocking potencies on epinephrine concentration-response curves. RESULTS: By means of RT-PCR technique alpha(1a)- and alpha(1b)-adrenoceptor transcripts were detected in the HUV. The blocking potency values of RS-100329 or B8805-033 against responses mediated by epinephrine were not consistent with the activation of an alpha(1A)-adrenoceptor population. Moreover, the low potency of the agonist A-61603 was not in accordance with an alpha(1A)-adrenoceptor interaction. On the other hand, the antagonist potencies of spiperone, AH11110A and cyclazosin were in agreement with an interaction on alpha(1B)-adrenoceptor subtype. CONCLUSION: Although alpha(1a)- and alpha(1b)-adrenoceptor messenger RNAs are detected in the HUV, only alpha(1B)-adrenoceptors are involved in epinephrine vasoconstrictor action.

Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein.

1. This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV). 2. HUV rings were mounted in organ baths and concentration-response curves to U-46619 (TXA(2) mimetic) were constructed in the absence or presence of SQ-29548 or ICI-192,605 (TP receptor antagonists). U-46619 was a potent constrictor (pEC(50): 8.03). SQ-29548 and ICI-192,605 competitively antagonized responses to U-46619 with pK(B) values of 7.96 and 9.07, respectively. 3. Concentration-response curves to EP receptor agonists: PGE(2), misoprostol and 17-phenyl-trinor-PGE(2) gave pEC(50) values of 5.06, 5.25 and 5.32, respectively. Neither pEC(50) nor maximum of PGE(2) and 17-phenyl-trinor-PGE(2) concentration-response curves were modified by the DP/EP(1)/EP(2) receptor antagonist AH 6809 (1 micro M). However, ICI-192,605 produced a concentration-dependent antagonism of the responses to all the EP receptor agonists. The pA(2) estimated for ICI-192,605 against PGE(2) or misoprostol were 8.91 and 9.22, respectively. 4. Concentration-response curves to FP receptor agonists: PGF(2)(alpha) and fluprostenol gave pEC(50) values of 6.20 and 5.82, respectively. ICI-192,605 (100 nM) was completely ineffective against PGF(2)(alpha) or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 micro M) against PGF(2)(alpha) was observed. 5. In conclusion, the findings obtained with TP-selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel.

Characterization of 5-HT receptor subtypes mediating contraction in human umbilical vein. 1. Evidence of involvement of 5-HT2A receptors using functional and radioligand binding assays.

This study attempted to characterize pharmacologically the involvement of 5-HT(2A) receptors in 5-HT-induced contractile responses in human umbilical vein (HUV) rings employing functional and radioligand binding assays. In HUV rings, prazosin 1 micro M did not affect contractile responses elicited by 5-HT, ruling out the involvement of alpha(1)-adrenoceptors in contractile responses to 5-HT. 5-HT-induced contractions were competitively blocked by ketanserin, a 5-HT(2A)-selective antagonist. The apparent pA(2) value was 9.8 and the Schild slope significantly less than unity, suggesting that 5-HT-induced responses are mediated by a heterogeneous receptor population. Alpha-methyl-5-HT, a selective 5-HT(2) receptor agonist, induced contractions that were antagonized in a competitive manner by ketanserin. The slope regression was not significantly different from unity and the pA(2) value was 8.8. The selective 5-HT(2A) ligand spiperone produced a parallel rightward shift on 5-HT CRCs in HUV rings. The calculated pA(2) was 9.0, which is in accord for an interaction with the 5-HT(2A) receptor subtype. Alpha-methyl-5-HT CRCs were competitively blocked by spiperone treatment. The Schild analysis yielded a pA(2) of 9.1 with a slope not significantly different from unity. The 5-HT(2C/2A) antagonist mesulergine 10 nM did not affect 5-HT CRCs, suggesting that 5-HT(2C) receptors are not involved in 5-HT-elicited contractions. Higher concentrations of mesulergine showed a parallel rightward shift on 5-HT responses. The calculated pA(2) was 7.44, which suggests an interaction with the 5-HT(2A) receptor subtype. In addition, mesulergine competitively blocked alpha-methyl-5-HT CRCs. The Schild slope was not significantly different from unity and the p A(2) value was 7.98. The binding of [(3)H]ketanserin to HUV membranes was saturable and of high affinity. Ketanserin displayed a monophasic curve which was best fit with a single component of binding. Nonlinear least squares analysis of the binding curves revealed a high affinity K(d) of 0.30 nM and a B(max) of 134 fmol/mg protein. These findings provide strong pharmacological evidence of the involvement of 5-HT(2A) receptors in 5-HT-induced vasoconstriction in HUV. In addition, the contribution of another receptor population cannot be excluded. The results also suggest that this receptor population is neither an alpha(1)-adrenoceptor nor a 5-HT(2C) receptor subtype.

Characterization of 5-HT receptor subtypes mediating contraction in human umbilical vein. 2. Evidence of involvement of 5-HT1B receptors using functional studies.

Previous studies have shown that a heterogeneous 5-HT receptor population may be involved in vasoconstrictor actions of 5-HT in human umbilical vein (HUV). The aim of the present study was to evaluate whether the 5-HT(1B/1D) receptor subtype mediates contraction in this tissue. 5-HT(1B/1D)-mediated responses can be enhanced or unmasked after exposure to threshold or sub-threshold KCl concentrations. In HUV rings, when 5-HT, alpha-Me-5HT or bradykinin concentration-response curves (CRC) were generated in the presence or absence of sub-threshold concentrations of KCl, there were not significant differences between the control and the treated rings. On the other hand, sumatriptan, the classic selective 5-HT(1B/1D) receptor agonist, produced a concentration-related contraction that was potentiated in the presence of sub-threshold KCl concentration. In addition, L-694,247, the novel selective 5-HT(1B/1D) receptor agonist, displayed a concentration-dependent contraction with high potency in HUV. The presence of sub-threshold concentrations of KCl produced a marked leftward shift of its CRCs.GR-55562, a 5-HT(1B/1D)-selective antagonist, competitively blocked sumatriptan CRCs with an estimated p A(2) of 8.00 and a slope not different from unity. Likewise, SB-216641, a selective 5-HT(1B) antagonist, produced a parallel rightward shift of sumatriptan CRCs in HUV. The Schild analysis yielded a p A(2) of 9.29, with a slope not different from unity. In addition, L-694,247 contractile responses were competitively blocked by SB-216641 with an estimated p A(2) value of 9.12 and a Schild slope not different from unity. On the other hand, ketanserin behaved as a weak antagonist of L-694,247-induced responses, yielding a calculated p A(2) value of 6.40. In summary, the results obtained in this study support that the 5-HT(1B) receptor subtype is involved in vasoconstrictor responses in HUV.

Further pharmacological evidence of nuclear factor-kappa B pathway involvement in bradykinin B1 receptor-sensitized responses in human umbilical vein.

Bradykinin (BK) B(1) receptors are thought to exert a pivotal role in maintaining and modulating inflammatory processes. They are not normally present under physiological situations but are induced under physiopathological conditions. In isolated human umbilical vein (HUV), a spontaneous BK B(1) receptor up-regulation and sensitization process has been demonstrated. Based on pyrrolidine-dithiocarbamate inhibition, it has been proposed that this phenomenon is dependent on nuclear factor-kappaB (NF-kappaB) activation. The aim of this study was to further evaluate the NF-kappaB pathway involvement on BK B(1) receptor sensitization in isolated HUV, using several pharmacological tools. In 5-h incubated rings, either the I-kappaB kinase inhibitor 3-(4-methylphenylsulfonyl)-2-propenenitrile (Bay 11-7082) or the proteasome activity inhibitor Z-Leu-Leu-Leu-CHO (MG-132) inhibited the development of the BK B(1) receptor-sensitized contractile responses. Furthermore, pro-inflammatory cytokine interleukin-6 (IL-6) produced a leftward shift of the concentration-response curve to the BK B(1) receptor agonist, whereas anti-inflammatory cytokines interleukin-4 (IL-4) and tumor growth factor-beta1 (TGF-beta1) produced a rightward shift of the responses to des-Arg(9)-BK in our preparations. Taken together, these results point to NF-kappaB as a key intermediary in the activation of the expression of BK B(1) receptor-sensitized responses in HUV and support the role of inflammatory mediators in the modulation of this process.